A two-year experimental program is proposed, in response to PA-06-418 Exploratory/ Developmental Bioengineering Research Grants, to study in vitro growth of early embryonic cardiac myocytes in a novel and uniquely suitable culture geometry, artificial myocardial tubes under normal or reversed transmural strain gradients. Our principal hypothesis is that uneven physical strain within the heart wall and resultant conditioning are physical cues critical to early maturation of cardiac muscle. Aim 1 will elucidate effects of uneven wall strain upon maintenance or stimulation of cellular proliferation or, conversely, commitment to a non-dividing, fully differentiated state. We will challenge both control and everted donuts, assembled into in vitro tubes, with diverse pressure stimuli and follow cytokinetic indices of proliferation, differentiation and cell death. We will measure opening angles of cultured in vitro loops to test the hypothesis that residual wall strain of everted donuts, demonstrably higher at time zero, will return toward normal as remodeling takes place. Aim 2 will explore molecular changes associated with conditioning paradigms established in Aim 1. We will use histochemical, immunohistochemical and proteomics approaches to 1) characterize kinetics of specific candidate molecules (growth factors, cytoskeletal and matrix molecules) and 2) discover new candidates of potential relevance to the cellular decision to differentiate and stop - or to resume - cell cycling. Aim 3 will systematically optimize growth and vigor of in vitro myocardial tubes, co-culturing them with epicardial or neural crest donor tissues. Growth and cytokinetics will be studied as in Aim 1 and molecular changes explored as in Aim 2. Significance of this innovative program of experiments lies in further understanding of physical contributions to regional growth and differentiation of embryonic cardiac muscle, conduction tissues, and to maturation and hypertrophic responses of the entire heart wall. These studies should also contribute to novel strategies for engineering muscle constructs for regenerative therapies and for encouraging non- proliferating myocytes to regain proliferative capacity. PUBLIC HEALTH RELEVANCE: Relevance of this project to public health lies in understanding of physical contributions to the embryonic formation and malformation of the heart and other muscle, to regional growth of cardiac conduction tissues, and to maturation of the heart wall, in health and disease. These studies should also contribute to novel strategies for engineering muscle constructs for regenerative therapies and for encouraging non-dividing muscle to regain proliferative capacity.